The ATP-binding cassette (ABC) superfamily is one of the largest and most highly conserved class of integral membrane proteins and is involved in the ATP-dependent transport of solutes across cellular membranes. Prominent members in this family include P-glycoprotein linked to multidrug resistance to chemotherapy for cancers and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a chloride channel whose malfunction causes cystic fibrosis, the most common lethal genetic disease in Caucasians. One unique feature of CFTR is that it has two distinct nucleotide binding domains (NBDs) that play critical roles in the gating of CFTR channels. Our understanding of the molecular basis of the regulation mechanisms, however, remains primitive. Unresolved questions include: What is the functional role of the individual NBD? Which NBD opens the channel, and which NBD closes the channel? How do the two NBDs interact with each other? Do they form a dimeric structure? If yes, does this dimeric structure depend on the state of the channel? These are the fundamental questions that interest a broad spectrum of biologists. A quantitative, multi-disciplinary approach will be used to tackle the molecular mechanisms whereby CFTR gating is regulated. It is a combination of structural modeling, energetic studies, patch-clamp recordings, biochemical assays and chemical synthesis. We plan to investigate quantitatively the functional roles of NBDs in the gating of CFTR channels. The two parallel aims in this project are: Aim 1. To engineer the nucleotide-binding pockets of CFTR to distinguish the functional roles of the NBDs. Aim 2. To investigate the hetero-dimeric structure of the NBD complex. A clear understanding of the quantitative mechanisms of the functions of CFTR is essential to future therapeutic design for CFTR-related diseases such as cystic fibrosis and secretory diarrhea. The methods as well as the results are directly applicable; to quantitative structure-function studies on other ABC transporter proteins.